This invention relates to nucleic acid and amino acid sequences of a novel human prostate-associated protease and to the use of these sequences in the diagnosis, prevention, and treatment of gastrointestinal disorders, cancer, and prostate disorders.
Prostate-specific antigen (PSA) is a 33 kD glycoprotein synthesized in the epithelial cells of the prostate gland. It is a secreted serine protease of the kallikrein family. PSA has been shown to digest the seminal vesicle protein, semenogelin, parathyroid hormone-related protein, and insulin-like growth factor-binding protein-3 (Henttu P. et al. (1994) Ann. Med. 26: 157-164; Cramer S. D. et al. (1996) J. Urol. 156: 526-531).
Genes encoding the three human kaillkreins, tissue kallikrein (KLK1), glandular kallikrein (KLK2), and PSA, are located in a cluster at chromosome map position 19q13.2-q13.4 (Riegmen P. H. (1992) Genomics 14:6-11). PSA shares more extensive homology with KLK2 than with KLK1. Both PSA and KLK2 are produced by prostate epithelial cells, and their expression is regulated by androgens. Three amino acid residues were found to be critical for serine protease activity, residues H65, D120, and S213 in PSA (Bridon D. P. et al. (1995) Urology 45: 801-806). Substrate specificity, described as chymotrypsinogen-like (with KLK2) or trypsin-like (with PSA), is thought to be determined by S207 in PSA and D209 in KLK2 (Bridon et al., supra). KLK1 is chymotrypsinogen-like and expressed in the pancreas, urinary system, and sublingual gland. KLK1, like the other kallikreins, is made as a pre-pro-protein and is processed into an active form of 238 amino acids by cleavage of a 24 amino acid terminal signal sequence (Fukushima D. et al. (1985) Biochemistry 24: 8037-8043).
Adenocarcinoma of the prostate accounts for a significant number of malignancies in men over 50, and over 122,000 new cases occur per year in the United States alone. Prostate-specific antigen (PSA) is the most sensitive marker available for monitoring cancer progression and response to therapy. Serum PSA is elevated in up to 92% of patients with prostatic carcinoma, and serum concentration depends upon tumor volume. Since PSA is also moderately elevated in patients with benign prostate hyperplasia, additional techniques are needed to distinguish between the two.
The enterokinases (also called enteropeptidases) are a functionally distinct family of serine proteases with homology to PSA and the kallikreins. Enterokinases act in a multi-step enzymatic cascade that allows the digestion of exogenous macromolecules without destroying similar endogenous material. This cascade results in the conversion of pancreatic proenzymes to active enzymes in the lumen of the gut. Trypsin, chymotrypsin, and carboxypeptidase A are examples of pancreatic enzymes activated by intestinal enterokinases, Enterokinase has a high specificity for the amino acid sequence (Asp)4-Lys, a motif found in the amino-termini of trypsinogens from a wide range of species. Congenital deficiency in enterokinase may cause life-threatening intestinal malabsorption.
The catalytic subunit of bovine enterokinase was cloned and characterized by LaVallie E. R. et al. (1993, J. Biol. Chem. 268: 23311-23317). The bovine enterokinase is a serine protease with four predicted intramolecular disulfide bonds and shares homology with other serine proteases, such as the kallikreins and hepsin. Like the kallikreins, bovine enterokinase has characteristic active site histidine, aspartic acid, and serine residues at conserved positions.
Discovery of proteins related to PSA, enterokinase, and the polynucleotides encoding them satisfies a need in the art by providing new compositions useful in diagnosis, prevention, and treatment of gastrointestinal disorders, cancer, and prostate disorders,
The present invention features a novel human prostate-associated kallikrein, hereinafter designated HUPAP, and characterized as having chemical and structural similarity to serine proteases such as bovine enterokinase, human pancreatic kallikrein, and rat renal kallikrein.
Accordingly, the invention features a substantially purified HUPAP which has the amino acid sequence shown in SEQ ID NO:1.
One aspect of the invention features isolated and substantially purified polynucleotides that encode HUPAP. In a particular aspect, the polynucleotide is the nucleotide sequence of SEQ ID NO:2.
The invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2 or variants thereof. In addition, the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2.
The invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode HUPAP. The present invention also features antibodies which bind specifically to HUPAP, and pharmaceutical compositions comprising substantially purified HUPAP. The invention also features the use of agonists and antagonists of HUPAP. The invention also features a method for producing HUPAP using the host cell, and methods for treating cancer and prostatic hyperplasia by administering an antagonist to HUPAP. The invention also features methods for treating gastrointestinal disorders by administering HUPAP or an agonist to HUPAP. In addition, the invention features methods for treating cancer and prostate disorders by administering an antagonist to HUPAP.